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-   -   Help with RT-PCR, late curves (http://www.molecularstation.com/forum/real-time-pcr-quantitative-pcr-forum/86424-help-rt-pcr-late-curves.html)

dialsaleem 09-12-2012 08:29 PM

Help with RT-PCR, late curves
 
Hi All,
I am new to the forum and RT-PCR. In my experiment the HK gene amplifies at 30 cycles but my target gene starts at 40-45, which I think is quite late.
What should I do? I have tried different concentrations of cDNA and repeated the RT on the samples.
The target gene is expressed low in the tissue, according to the litreature.
Is the data obtained after 30 cycles still usefull?
Can anyone suggest another Housekeeping gene which starts amplifying, lets say after 35-40cycles?
Thanks in advance,

namishu95 09-17-2012 06:13 PM

Re: Help with RT-PCR, late curves
 
is it human HK gene? which one?, we are using RP assay for detection of Human RNase P, we usually got around middle of 30 of Ct value from original human nasal swap specimens, is it what you are looking for?

butters 09-18-2012 12:53 AM

Re: Help with RT-PCR, late curves
 
Importantly have you run a gel of the said 40-45 cycles (gel to show correct size amplification) or the amplification is based on peak on the RT system?

Sira 09-18-2012 04:11 AM

Re: Help with RT-PCR, late curves
 
Hello,what's the reagents you're using?


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